1. Field of the Invention
The field of this invention relates to a solid phase system for performing an immunoassay for detection and quantitation of an analyte suspected of being in a specimen. More particularly, the invention relates to a method using a solid support surface consisting of particles to which can be bound a binding component or antigen.
2. Prior Art
Numerous methods utilize immunoassay techniques for more precise and reliable ways to quantitate an amount of analyte in a specimen. The rapid, quantitative and cost-efficient results desired by the medical and diagnostic testing industry have provided the impetus for novel means of accomplishing these goals. There are two types of assay systems currently used. One is a homogeneous system whereby the assay is performed in a single phase. Antibody and analyte are allowed to react together and a labelled material is introduced which binds to the antibody or antigen after an antibody:antigen complex has formed. The label generates a signal which is readable when present in the reaction zone or solution. The signal is correlated with the amount of analyte present in solution. This indirect measurement is generally used because of the difficulty of direct measurement of many analytes.
The second type of system is a heterogeneous assay which is a two-phase system where there is a solid, or bound, phase and a liquid, or an unbound phase, requiring an additional step to separate the bound from unbound material. Typically, a solid support surface is used as the bound phase, to which is attached an antibody or antigen via a chemical bond or adsorption. Various types of solid support surfaces have been developed to improve the efficiency of the immunological reaction between antibody and antigen, and, to increase the efficiency of the separation step. An inefficient or incomplete separation of bound from unbound can result in unbound label remaining in the reaction zone after the separation step, which will cause a positive signal to be read where there is no antigen present. An efficient binding and subsequent separation are two of the most important aspects of a desirable assay.
Particles have been used in agglutination assay procedures for some time in order to overcome the drawbacks of inefficient binding. In this type of system soluble antigens will combine with their specific antibody to form a precipitate, in which the antigen-antibody complexes form large aggregates which are insoluble. The same antigens, if attached to particulate matter such as latex particles or bacterial cells, will form agglutinates or clumps. The agglutination reaction can be detected and quantified using visual or instrumentation means such as light-scattering or absorption techniques (Bellanti, "Immunology II", W. B. Saunders Company, Philadelphia, 1978, p. 212). Though the test is commonly used in the clinical laboratory, it suffers from several limitations, such as serum interferences, insensitivity, and, most importantly, from the perspective of the technician or scientist performing the test, the subjective judgment of the assay endpoint. While the method is convenient for qualitative analysis, it is inadequate for quantitative analytsis, especially of very low concentrations of analysis. Latex particles have been used as labels for the analyte of interest, whereby the assay relies on the use of an agglutination reaction to decrease the number of particles of a particular size present in the assay mixture (Boguslaski, et al., "Clinical Immunochemistry," Little, Brown and Company, Boston, 1984, p.211).
Various materials have been used as support surfaces, including glass rods, glass beads, silica impregnated strips, glass fiber, filter paper, cellulose or its derivatives, SEPHAROSE beads, plane polymeric surfaces, magnetizable solid phase cellulose/iron oxide particles, ferratin, etc.
Coated test tubes and trays have the limitation that only the inner surface of the vessel is coated with the solid phase. Material in the center of the solution will not be in intimate contact with the solid phase until and unless agitated; and even then only over a comparatively long period of time. The lack of surface area prevents the rapid establishment of an equilibrium between the bound and unbound phase.
Physical separation of the bound phase from the unbound phase is required in heterogeneous assays. Most often the bound phase is retained for measurement. Separation is generally accomplished by one of several methods including chromatography, filtration, electrophoresis, adsorption, precipitation, or centrifugation. It is preferable to use a method which is adaptable for use in automated equipment or in a system that can be used by a trained technician.
Thus, Michael E. Jolley, Ph.D., discloses in Pandex Laboratories Research Report No. 1, July 1983, a particle concentration fluorescence immunoassay wherein 0.6-0.8 um polystyrene particles are bound to the antigen of interest. A first antibody directed against the antigen and a labelled second antibody directed against the first antibody are contacted with the bound antigen. The label is limited to a fluorescent molecule and is read after separation by front-surface fluorimetry.
Such procedure contains deficiencies in that it is limited to fluorescent signal and detection means and it is also limited to front-surface reading; which does not appear to be pragmatic for rear surface viewing. Moreover, the particles disclosed are only composed of polystyrene and 0.6-0.8 um in size, rather than a broad range of useful particle compositions and sizes.
U.S. Pat. No. 4,201,763, issued to Monthony, et al., discloses water insoluble hydrophilic polymeric particles in the performance of a sandwich or competitive immunoassay. The method is limited to the use of a fluorescent label and therefore lacks the versatility of uses in situations where nonfluorescent labels are perferrable.
U.S. Pat. No. 4,415,700, issued to Batz, et al., describes hydrophilic latex particles consisting of a homo- or co-polymer of monomers containing at least one epoxy group and at least one polymerizable carbon-carbon double bond in the molecule. The method using the particles is a competitive assay wherein labelled first antibody bound analyte and unlabelled first antibody bound analyte compete for binding sites on a particle bound second (anti-first antibody) antibody. Again, the invention is limited to one type of label, in this instance an enzyme, and is also limited to a particular particle composition.
With the emergence of the physician's office and self-testing markets, there is a need for a separation technique that can be used by relatively unskilled users. A solid phase that would be compatible with an easy-to-use separation system, that provided accurate and repeatable results would be advantageous. Such a method should have a solid phase that permits a rapid equilibrium to be established between the antibody and antigen so as to promote rapid binding of the two; it should also be convenient and inexpensive; and, it should be usable in settings in addition to a hospital or laboratory, i.e., the physician's office or in the home.